The invention consists in providing specific DNA sequences and selecting DNA regions which are particularly suitable for detecting bacteria. Thus this application is based on the identification of organisms by their genetic information. Using deviations of as little as a single component in the nucleotide sequence in certain DNA regions it is already possible to differentiate species.
Historically considered, ribosomal RNA genes have already been used for phylogenetic classification of organisms. Comparisons of sequences of the 5 S and 16 S ribosomal genes in different bacteria have led to significant corrections in assignments of relatedness and to discovery of the kingdom of the Archaebacteria. Because of its size and the corresponding high sequencing effort, 23 S RNA has only in recent years been used for systematic classifications.
Direct sequencing of genes of microorganisms to be identified was too expensive and time-consuming in practical use. In the 1980s, therefore, specific nucleotide probes were used to detect bacteria. While those can show very good specificity, the detection limit is often too low. The probe technology was substantially improved by combination with amplification techniques, which reproduce the nucleotide sequence to be detected and thus substantially increase the sensitivity of detection. In an extreme case, it is possible to detect a single isolated genome. In practice, losses occur in isolation of DNA, increasing the detection limit to about 102 to 104 cells.
On the basis of fundamental research, DNA probes from the 5 S, 16 S and 23 S genes were utilized for practical applications. For instance, one should note these patents: Nietupski et al. (U.S. Pat. No. 5,147,778) for detection of Salmonella; Mann and Wood (U.S. Pat. No. 6,554,144) for detection of Yersinia species; Leong (EP 04 79 117 A1) for detection of various Gram negative and Gram positive bacteria; Carico et al. (EP 1 33 671 B1) for detection of various enterobacterial species; Shah et al. (EP 03 39 783 B1) for detection of Yersinia enterolytica; Carrico (EP 01 63 220 B1) for detection of Escherichia coli; Hogan et al. (WO 88/03957) for detection of species of Enterobacteria, Mycobacterium, Mycoplasma and Legionella; Leiser et al. (WO 97/41253) for detection of various microorganisms; Grosz and Jensen (WO 95/33854) for detection of Salmonella enterica; Stackebrandt and Curiaie (EP 03 14 294 A2) for detection of Listeria monocytogenes; Wolff et al. (EP 04 08 077 A2), Hogan and Hammond (U.S. Pat. No. 5,681,698) for detection of Mycobacterium kansasii; Hogan et al. (U.S. Pat. No. 5,679,520) for detection of various bacteria; Kohne (U.S. Pat. No. 5,567,587) particularly for detection of bacterial RNA; Kohne (U.S. Pat. No. 5,714,324) for detection of various bacteria; Pelletier (WO 94/28174) for detection of Legionella; and Kohne (U.S. Pat. No. 5,601,984) for detection of various bacteria. Most of the patents relate to the sequence of the 16 S rDNA gene, and many also relate to the 23 S rDNA.
It appeared, though, that the latter genes are not suitable for many differentiation operations in practical use because they are too strongly conserved. Closely related microorganisms in particular cannot be differentiated. On the other hand, the 5 S rDNA gene is generally too variable and its differentiation potential is too low for practical use, even though it was initially used for phylogenetic studies in basic research because of its small size.
As the 5 S, 16 S and 23 S rDNA genes have many disadvantages as diagnostic aids, DNA regions which could be used for identification of all eubacteria were sought. Such a DNA region should have very variable and, at the same time, strongly conserved sequences. Then the variable regions would be useful to differentiate closely related species, such as strains and species. The conserved sequences would be used to detect more distantly related bacteria or higher taxonomic units.
In the very recent past, the 16 S–23 S transcribed spacer has been discussed in the literature in the context of extensive studies on ribosomal operons. Their applicability in systematic bacteriology has been questioned, though. For example, Nagpal et al. (J. Microbiol. Meth. 33, 1998, p. 212) considered the utility of these spacers very critically: A major problem with this transcribed rDNA spacer is that it frequently contains tRNA insertions. Such insertions represent dramatic changes in the sequences, and do not necessarily have a relation to phylogenetic separations. However, they have been used in the past to utilize the length polymorphism which they cause as a phylogenetic characteristic (Jensen et al. 1993, Appl. Envir. Microb. 59, 945–952; Jensen, WO 93/11264; Kur et al. 1995, Acta Microb. Pol. 44, 111–117).
The transcribed spacer between the 23 S and 5 S rDNA is an alternative target sequence for identification of bacteria. For instance, Zhu et al. (J. Appl. Bacteriol. 80, 1996, 244–251) published detection of Salmonella typhi using this diagnostic DNA region. However, the general utility of this spacer for detecting other bacteria cannot be derived from that work. There are very many examples which indicate that a DNA region is suitable only for identifying one or a few species of bacteria. Individual patents imply a potential but very limited applicability of the 23 S–5 S transcribed DNA region for bacterial diagnosis. Those all have in common that their applicability is limited to just a single bacterial species, specifically, to detection of Legionella (Heidrich et al., EP 07 39 988 A1), Pseudomonas aeruginosa (Berghof et al., DE 197 39 611 A1) and Staphylococcus aureus (Berghof et al., WO 99/05159).
The technical problem underlying the present invention consists in providing materials and processes which allow to detect any desired bacterium (preferably from the Enterobacteria group) in a material being examined.
This problem is solved according to the invention by a nucleic acid molecule as a probe and/or a primer for detection of bacteria, selected from                a) a nucleic acid comprising at least one sequence with any of the SEQ ID NOs: 1 to 530 and/or a sequence from position 2667 to 2720, 2727 to 2776, 2777 to 2801, 2801 to 2832, 2857 to 2896, 2907 to 2931, 2983 to 2999, and/or 3000 to 3032 according to SEQ ID NO: 1; or nucleic acids homologous with them;        b) a nucleic acid which hybridizes specifically with a nucleic acid according to a);        c) a nucleic acid which exhibits 70%, and preferably at least 90%, identity with a nucleic acid according to a) or b);        d) a nucleic acid which is complementary to a nucleic acid according to any of a) to c);        and/or        combinations of the nucleic acids according to any of a) to d), except for the SEQ ID NO:1.        
Further claims concern preferred embodiments.
In one particularly preferred embodiment, the presence of Enterobacteria in a sample being analyzed is shown by the analysis sample being brought into contact with a probe which detects the presence of a nucleic acid from the 23 S/5 S rDNA genome segment of the Enterobacteria.
The sequence specified as NO: 1 in claim 1 is derived from E. coli. Homologous DNA sequences are those derived from bacteria other than the E. coli sequence shown, but in which the genome segment from the other bacteria corresponds to the sequence based on SEQ ID NO:1. For more details, we refer to the definition of homologous DNA sequences, below.
The nucleic acid molecule according to the invention comprises preferably at least 10 nucleotides, and especially preferably at least 14 nucleotides. Nucleic acid molecules of these lengths are used preferably as primers, while nucleic acids used as probes preferably comprise at least 50 nucleotides.
In another preferred embodiment, nucleotides of the probe or the primer can be replaced by modified nucleotides containing, for instance, attached groups which ultimately are used for a detection reaction. Particularly preferred derivatizations are specified in claim 4.
In another preferred embodiment, combinations of the specified nucleic acid molecules are used. Selecting the particular combination of nucleic acid molecules allows adjustment of the selectivity of the detection reaction. In doing so, selection of the primer combinations and/or probe combinations can establish the conditions of the detection reactions so that they either demonstrate generally the presence of bacteria in a sample, or specifically indicate the presence of a certain bacterial species.
A kit according to the invention contains at least one nucleic acid according to the invention together with the other usual reagents used for nucleic acid detection. They include, among others, suitable buffers and detection agents such as enzymes with which, for example, biotinylated nucleic acid hybrids which are formed can be detected.
In another preferred embodiment, called Consensus PCR here, the process is carried out according to claim 8. First, a nucleic acid fragment is amplified by use of conserved primers (those hybridize to nucleic acids of different bacterial taxonomic units). Then more specific nucleic acid segments are detected by use of other more specific nucleic acids (these hybridize with only a few taxonomic units or only with a certain species). The latter allow then a conclusion about the presence of a particular genus, type or species in the sample being analyzed.
Various established detection procedures can be employed to detect nucleic acids in the process used. They include Southern Blot techniques, PCR techniques, LCR techniques, etc.
In one broad study, transcribed spacer between 23 S and 5 S rDNA was examined for its general usefulness as a diagnostic target molecule. For this purpose, genomic DNA from very many bacterial strains was isolated, purified, cloned into a vector, sequenced, and finally evaluated in an extensive sequence comparison. Surprisingly, this sequence segment was suitable for identification of almost all bacterial species. With the encouragement of that finding, the analyses were extended to the adjacent regions of the spacer. All in all, DNA fragments from all bacterial classes or smaller phylogenetic units were examined. They have lengths of 400–750 base pairs and include the end, i.e., the last 330–430 nucleotides (depending on the species) of the 23 S rDNA gene, the transcribed spacer, and the complete 5 S rDNA gene. The total size of the fragments is 400–750 base pairs. The experiments showed that the 23 S rDNA gene and the 5 S rDNA gene are adjacent in almost all bacterial species. This information is an important prerequisite for use and applicability of this invention.
This invention is particularly based on the fact that a DNA region which can contain significant portions of at least two adjacent genes is selected for detection of microorganisms. In practice, the usefulness of the region is determined particularly by its phylogenetic variability. There can be quite contrary requirements, depending on whether distantly related bacteria, taxonomic units, or strains of a species are to be detected. Now the frequency of occurrence of both variable and conserved regions is greater for two genes than for one, as the example of the 23 S–5 S tandem shows. Thus the use of two adjacent genes, including the variable intercalated sequences is a substantial advantage.
It was also found that the end of the 23 S rDNA gene, the 5 S rDNA gene, and the transcribed spacer between them contain nucleotide sequences which cover a wide range from very variable to very conserved. A fine analysis of this region provided further very interesting conclusions about the differentiation potential of various phylogenetic bacterial units (FIG. 2, Table 6). Nearly all taxonomic units can be detected and/or differentiated by using subregions. More or less variable regions are shown in FIG. 2 with the sections 1–9, while the strongly conserved regions are intercalated between and adjacent to them. The latter are thus particularly suitable for detecting higher taxonomic units, such as the whole Eubacteria or classes or divisions of them.
The phylogenetic dendrogram in FIG. 1 provides another indication of the usefulness of the region. It can be seen that the 23 S rDNA–5 S rDNA region allows very good differentiation with respect to coarse classification, as members of the Proteobacteria are assigned to 1–2 groups, while the Firmicutes are separated. Furthermore, the lengths of the branches, even for closely related species, indicates that they can be distinguished well from each other. Here a phylogenetically correct assignment of close relatives in the dendrogram is quite undesirable, because then they would lie in a closely connected coherent group and perhaps could not be distinguished as easily from one another.